J. Phys. Chem. B 2005, 109, 5259-5266
5259
Theoretical Study of General Base-Catalyzed Hydrolysis of Aryl Esters and Implications for Enzymatic Reactions Daiqian Xie,† Dingguo Xu,‡ Lidong Zhang,† and Hua Guo*,‡ Department of Chemistry, Institute of Theoretical and Computational Chemistry, Nanjing UniVersity, Nanjing 210093, People’s Republic of China, and Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131 ReceiVed: February 3, 2005
In this work, the mechanism of general base-catalyzed hydrolysis of aryl esters is investigated in vacuo with density functional theory and in solutions with a polarized continuum model. The hydrolysis is found to proceed via a concerted mechanism featuring simultaneous addition and elimination steps accompanied by proton transfers, consistent with experimental evidence. Reasonable agreement with measured kinetic isotope effects provides additional validation. It is found that solvation substantially lowers the transition state energy, but has a small effect on the reaction exothermicity. An enzyme oxyanion hole, modeled by an ammonia molecule hydrogen bonded to the acyl carbonyl oxygen, is found to stabilize the near-tetrahedral transition state. Implications of these findings for the hydrolysis step of the dehalogenation reaction catalyzed by 4-chlorobenzoyl-CoA dehalogenase are discussed.
I. Introduction The hydrolysis of esters is an important and prevalent class of reactions that have received a great deal of attention in both organic chemistry1-3 and biochemistry.4-6 While the reaction mechanism in solution is reasonably well-understood, many questions still remain to be answered for enzyme-catalyzed hydrolysis. The study of enzymatic hydrolysis is of great interest because the rate enhancement can be quite dramatic.7 The catalytic efficiency of these enzymes is thought to derive from their abilities to stabilize the transition state utilizing various strategies, such as active-site electrostatic interactions and oxyanionic hydrogen bonding.4,6,8-11 Detailed knowledge of the effects of these interactions will greatly advance our understanding of the catalytic power of enzymes. There are many examples of enzyme-assisted ester hydrolysis. In hydrolysis catalyzed by serine proteases,8,9 for instance, an enzyme-substrate ester intermediate is first formed in the acylation step via nucleophilic attack by a serine residue, a member of the catalytic triad (Ser-His-Asp). The nucleophilicity of the serine hydroxyl group is enhanced by a proton transfer to a nearby histidine, which is in turn activated by the aspartate. The subsequent cleavage of the ester bond (the deacylation step) is realized by reacting with a water nucleophile activated by a general base, namely, the imidazole group of the His residue. A particularly important instrument used by the enzyme to stabilize the transition state is an oxyanion hole through hydrogen bonding with the acyl carbonyl oxygen, where negative charge develops during the nucleophilic attack. The barrier is estimated to be lowered by a few kcal/mol.12-14 A similar hydrolysis strategy is employed by several dehalogenases,15 in which the ester is formed between the substrate and the carboxylate of an Asp residue in the proceeding substitution * Author to whom correspondence should be addressed. E-mail:
[email protected]. † Nanjing University. ‡ University of New Mexico.
step. Again, the nucleophilic attack by a water molecule is assisted by a His residue, and the hydrolysis transition state is also stabilized by an oxyanion hole. It is generally accepted that the hydrolysis of alkyl esters occurs via a stepwise addition-elimination mechanism.1 In the specific base-catalyzed hydrolysis, the attack of a hydroxide ion at the acyl carbonyl carbon results in a stable oxyanionic tetrahedral intermediate (TI), which subsequently collapses to form the products. The mechanism for general base-catalyzed hydrolysis, which is probably more relevant to enzymatic reactions,4 is similar, although the initial nucleophilic attack is accompanied by proton transfer from the water nucleophile to the general base.3 Accumulation of the TI has never been directly observed, but indirect experimental16-18 and theoretical evidence19-31 of its existence is quite convincing. For a good leaving group such as an aryloxide ion, there is overwhelming evidence that suggests that the hydrolysis proceeds via a concerted mechanism, in which the attack of the nucleophile and departure of the leaving group occur simultaneously.32-37 In fact, transition state analogues with tetrahedral geometries have been successfully designed to elicit antibodies that can efficiently catalyze the hydrolysis of many aryl esters.38 Although several high-level theoretical studies have recently been reported for the hydrolysis of alkyl esters,22,24-27,31 similar work on aryl esters is scarce.39-42 For example, Zheng and Bruice used PM3 to study the hydrolysis of CH3-CO-O-PhCO-SCH3 and found a marginally stable TI.40 More recent ab initio41 and density functional theory (B3LYP/6-31+G*) studies42 of phenyl acetate hydrolysis also suggested an unstable TI. However, none succeeded to locate a truly concerted transition state. In this work, we focus on the general base-catalyzed hydrolysis of aryl esters. Besides interests in understanding the mechanism in the gas phase and in solutions, we also discuss the reaction in the context of hydrolytic dechlorination of 4-chlorobenzoate (4-CBA) catalyzed by 4-CBA-CoA dehalogenases.43,44 This enzymatic dehalogenation reaction has at-
10.1021/jp0506181 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/11/2005
5260 J. Phys. Chem. B, Vol. 109, No. 11, 2005
Xie et al.
SCHEME 1 : Mechanism for Dehalogenation of 4-CBA-CoA Catalyzed by 4-CBA-CoA Dehalogenase
tracted much recent attention40,45-57 partly because of its practical implications in bioremediation of notorious halogenated aromatic pollutants such as the polychlorinated biphenyls (PCBs)58,59 and partly because of the unique catalytic mechanism of the enzyme. As depicted in Scheme 1, the covalent catalysis is initiated by nucleophilic attack on the benzoate C(4) position by the carboxylate group of Asp145,48 assisted by polarization of the benzoyl carbonyl oxygen by two backbone amide NH groups.46,49,51,53 The cleavage of the Cl-C(4) bond results in a detectable intermediate in which the substrate is covalently bonded with the enzyme.45,50 This arylated enzyme (EAr) complex is subsequently hydrolyzed by a water molecule activated by His90 to produce 4-hydroxybenzoyl-CoA (4HBA), which is eventually expelled by the enzyme. The catalytic role of the general base is evidenced by a significant reduction of the turnover rate constant kcat in the H90Q mutant.50 Theoretical studies of the enzymatic dehalogenation reaction have so far concentrated on the substitution step leading to the EAr intermediate.40,55-57 In particular, we have recently reported quantum mechanical/molecular mechanical (QM/MM)60-63 studies of the nucleophilic aromatic substitution (SNAr) reaction catalyzed by the 4-CBA-CoA dehalogenase.56,57 Our theoretical studies indicate that strong hydrogen bond stabilization forces acting on the benzoyl carbonyl oxygen by the backbone amide NH groups of Gly114 and Phe64 result in the significant lowering of the rate-limiting transition state relative to the solution reaction and the formation of the enzyme-Meisenheimer complex. However, little work has been reported on the reaction pathway for the subsequent hydrolysis step in this dehalogenation reaction. Experimentally, it is difficult to control the initial conditions of the hydrolysis step in this multistep reaction. Indeed, the structure of the EAr intermediate, which serves as the starting point of the hydrolysis step, has not been determined experimentally. In addition, it is not easy to identify kinetic intermediates and to accurately define the microscopic rate constants in multistep enzymatic reactions. For this and other reasons, computational studies64 become an alternative and powerful tool to gain insight into the microscopic mechanism of the catalyzed reaction. To this end, it is important to establish the mechanism and energetics of the nonenzymatic reaction with a high-level quantum chemical theory, which provides a benchmark to compare with the corresponding enzymatic reaction. By doing so, key contributing factors in the enzyme catalysis may be uncovered. In addition, the structural and energetic information obtained in such studies is invaluable in calibrating semiempirical quantum methods, such as AM165 or PM3,66 to be used in QM/MM studies of the enzymatic reaction. In this publication, we report detailed density functional theory and semiempirical PM3 studies of general base-catalyzed
SCHEME 2 : Model Systems Used to Study General Base-Catalyzed Hydrolysis of Aryl Esters
hydrolysis of aryl esters in three models. Full geometry optimizations of the stationary states were performed to map out the reaction pathway. The influence of an enzyme oxyanion hole is explored by including an ammonia molecule that forms a hydrogen bond with the acyl carbonyl oxygen. Solvent effects are considered within the polarized continuum model. The implications of these computational results for the enzymatic reaction are also discussed. This paper is organized as follows. Section II briefly describes the theoretical methods used in the calculation. The results are presented and their implications for the enzymatic reaction are discussed in section III. A short conclusion is given in section IV. II. Computational Methods A. Model Construction. As depicted in Scheme 2, three models were constructed to identify the important factors that can influence the enzymatic reaction. They are comprised of a phenyl ester, a water molecule, and an imidazole. The ester represents the covalent EAr intermediate formed between the 4-CBA-CoA substrate and Asp145 of the 4-CBA-CoA dehalogenase, while the imidazole models the His90 side chain. The atomic labels in Scheme 2 are meant to establish a connection with the active-site residues of the 4-CBA-CoA dehalogenase. The first two models differ at the benzene C(1) position, where the thioester carbonyl (CO-S-CH3) group in model I is substituted by a methyl (CH3) group in model II. An oxyanion hole is simulated in model III by an ammonia molecule hydrogen bonded to the acyl carbonyl oxygen. Such a hydrogen bond has been observed in the X-ray structure of the enzymeproduct (EP) complex47 between the indole NH group of Trp137 and an Asp145 carbonyl oxygen and in our earlier molecular dynamics (MD) study of the EAr complex as well.57 In all the calculations, no restrictions on the atomic positions were enforced.
General Base-Catalyzed Hydrolysis of Aryl Esters
J. Phys. Chem. B, Vol. 109, No. 11, 2005 5261
Figure 2. Reaction pathway for model I obtained from DFT calculations. The energies and geometries are given in Tables 1 and 2.
Figure 1. Snapshot of the EAr‚Cl- complex from previous QM/MM simulations of the SNAr reaction catalyzed by the 4-CBA-CoA dehalogenase. The chloride ion will be replaced by a water molecule for the hydrolysis of the EAr intermediate. The hydrogen bond between the indole NH of Trp137 and the acyl carbonyl oxygen is indicated by a dashed line.
The initial geometries of these models were extracted from a snapshot of the EAr‚Cl- complex obtained from our earlier MD simulation.57 Such a snapshot is depicted in Figure 1. Note the hydrogen bond between the acyl carbonyl oxygen and the indole NH of Trp137, denoted in the figure by a dashed line. Since the water nucleophile in the hydrolysis step is postulated to take the position of the Cl- ion between the EAr complex and the imidazole of His90,57 we simply replaced the Cl- with H2O for our calculations. As described below, the exact snapshot used for the initial geometry is of no particular importance because it is subjected to a full geometric optimization. It should be made clear at the outset that these truncated models are not meant to give a quantitatively accurate representation of the active site. For example, the oxyanion hole consisting of the backbone NH groups of Gly114 and Phe64 was not included in these models. Thus, their influence via the benzoyl carbonyl cannot be examined. Nor is the backbone carbonyl CdO of Ala86. Engaged in hydrogen bonding with Nδ1-H of the His90 imidazole,50 its existence may increase the basicity of the general base. Nevertheless, such active-site models67 should provide a useful starting point to understand the hydrolysis mechanism in vacuo, in solution, and in the enzyme. B. Electronic Structure Calculations. All density functional theory (DFT) calculations presented here were carried out using the B3LYP functional,68,69 as implemented in the Gaussian 03 suite of quantum chemistry programs.70 The DFT approach was chosen because of its partial inclusion of electron correlation effects, which is expected to give a much better description of reaction barriers and hydrogen bonds than the Hartree-Fock method. The geometries of the stationary points, including the reactant complex (RC), the product complex (PC), and the transition state (TS), were fully optimized using the 6-31+G(d,p) basis set. The inclusion of diffuse functions in these calculations is necessary because of the involvement of anionic species. The stationary points were confirmed by additional frequency calculations, which also allowed the calculation of thermodynamic properties. The connectivity between the stationary points was established by intrinsic reaction coordinate (IRC) calculations.71,72 The CHelpG charges73 were calculated
Figure 3. Reaction pathway for model II obtained from DFT calculations. The energies and geometries are given in Tables 1 and 2.
at these stationary points. To test the convergence of the basis set, we have also carried out single-point calculations for model II using the 6-311+G(d,p), 6-31++G(d,p), and 6-311++G(d,p) basis sets. Since the reaction involves charge-separated species, solvation is expected to be important. Ideally, the optimization of solvated species should be performed with explicit solvents, as in some recent work.25,74,75 However, such optimization can be quite involved and in general not stable. As a result, we elect in this work to use an implicit solvent model. In particular, the polarized continuum model (PCM)76,77 was used at the geometries of the gas-phase stationary points without further optimization. The dielectric constants used in the calculations are ) 78.4 for water and 4.9 for chloroform. Chloroform was chosen because its dielectric constant is close to that in the protein interior.78 Additional calculations were carried out using the PM3 method,66 again with the Gaussian 03 program. The purpose here is to examine the performance of the semiempirical method in describing the general base-catalyzed hydrolysis and to establish its suitability for future free energy simulations of the enzymatic reaction using the QM/MM strategy. As in the DFT calculations, the corresponding stationary points were located by full geometry optimization. III. Results and Discussions The reaction pathway and geometries of the DFT stationary points are depicted in Figures 2-4 for the three models, respectively. Additional energy data, including the zero-point energy corrections and free energies, are summarized in Table 1. Key geometric information such as internuclear distances and bond angles are listed in Table 2, while atomic charges are provided in Table 3. The kinetic isotope effects (KIEs) are collected in Table 4. The PM3 results are discussed separately in section III.D, and the corresponding stationary point energies and geometries are listed in Table 5 and shown in Figure 5.
5262 J. Phys. Chem. B, Vol. 109, No. 11, 2005
Xie et al.
Figure 4. Reaction pathway for model III obtained from DFT calculations. The energies and geometries are given in Tables 1 and 2.
TABLE 1: Energetics (kcal/mol) for General Based-Catalyzed Hydrolysis in Vacuo (B3LYP/6-31+G(d,p)) and in Solutions (PCM)a models
a
TS
PC
I
energy ZPE free energy PCM(H2O) PCM(CHCl3)
32.47 32.07 36.43 16.14 23.28
-8.82 -8.03 -8.39 -9.63 -9.36
II
energy 6-31++G(d,p) 6-311+G(d,p) 6-311++G(d,p) ZPE free energy PCM(H2O) PCM(CHCl3)
37.15 37.14 37.88 37.83 37.01 44.28 16.05 27.60
-6.40 -6.46 -6.53 -6.54 -5.66 -4.46 -8.00 -6.63
III
energy ZPE free energy PCM(H2O) PCM(CHCl3)
34.66 35.45 39.30 21.78 27.49
-17.39 -15.23 -13.36 -13.63 -16.18
The energies are given relative to RC.
As shown in Table 1, the barrier height and exothermicity obtained for model II with the larger 6-311+G(d,p), 6-31++G(d,p), and 6-311++G(d,p) basis sets are almost quantitatively the same (
